Influenza A virus (IAV) strains, members of the orthomyxovirus family, are the leading cause of human death from respiratory diseases despite the existence of vaccine prophylaxis. Contributing factors are poor vaccine protection of the elderly and lowered vaccine efficacy when the circulating strains differ from those included in the seasonal vaccine. The problem is further aggravated through increasing compromise of licensed anti-IAV therapeutics by pre-existing or rapidly emerging viral resistance. Recognizing the unmet clinical need for a new generation of efficacious, readily applicable IAV therapeutics, it is the overarching goal of this proposal to apply a rigorous drug development approach to the problem and ultimately identify an anti- IAV therapeutic candidate and at least one mechanistically distinct alternative compound that are suitable for IND-enabling formal development. Narrowly focused drug discovery campaigns are at high risk of early stage failure. We hypothesize that a comprehensive anti-IAV approach interrogating the full host-pathogen interactome for pathogen-directed and host-directed drug candidates has the highest prospect of ultimately yielding viable clinical candidates. This is based on the realization that traditional pathogen-directed therapeutics enjoy an excellent clinical record but can be compromised by emerging viral resistance and/or a narrow pathogen indication spectrum, whereas host-directed antivirals promise to overcome these limitations, yet are at present predominantly still in preclinical development. Building on our multiple-year expertise in the development of myxovirus inhibitors, we have in pilot studies generated a replication-competent IAV recombinant expressing a luciferase reporter and developed a drug screening protocol that allows the simultaneous identification of pathogen-directed and host- directed antivirals. Proof-of-concept implementation of the approach has yielded, amongst others, a novel host- directed agonist class of cellular antiviral defense pathways with nanomolar anti-IAV activity. To maximize the prospect of success of this program, we will in the R21 phase diversify our current portfolio of anti-IAV candidates by implementing a large-scale drug screening campaign interfaced with orthogonal counterscreens (specific aim 1). Novel anti-IAV candidates and existing leads will be mechanistically classified and a subset of distinct lead candidates short-listed based on mechanistic class and defined potency and toxicity milestones in cell lines and primary cells (specific aim 2). Selected candidates will be subjected to a hit-to-lead synthetic optimization program guided by potency, ADME/tox, and pharmacokinetic parameters, and the molecular target identified (specific aim 3). Assessment of prophylactic and therapeutic efficacy, toxicokinetic properties, the effect of treatment on the dynamics of virus host invasion, virus shedding, and transmission profiling of resistant recombinants will drive the educated selection of a clinical candidate and mechanistically and structurally distinct alternative for formal pre-clinical development (specific aim 4).